Pollen Morphology of Convolvulaceae from Southeastern Amazonian Cangas and Its Relevance for Interaction Networks and Paleoenvironmental Studies

Serra dos Carajás harbors a unique open plant community in Amazonia, known as canga vegetation, with several endemic species coexisting with the potential threat of large-scale iron ore mining. In this sense, Convolvulaceae occur in a wide variety of canga geoenvironments with multiple flower visitors, but the scarcity of data on its pollen morphology prevents the correct association between Convolvulaceae species with floral visitors, as well as the precise identification of their habitats throughout the Quaternary. Therefore, this study aims to contribute to the taxonomic knowledge and refinement of the identification of insect-plant networks of endangered plants, including Ipomoea cavalcantei. Pollen grains were examined by light and scanning electron microscopy (LM and SEM, respectively), and the morphological parameters obtained were statistically analyzed using principal component analysis. Therefore, all species were differentiated based on aperture types and exine ornamentation. The set of morphological characters indicated that echinae morphology, easily identified under LM, was effective for the identification of Ipomoea species. This work represents the first robust pollen database for a precise identification at the species level of Convolvulaceae from southeastern Amazonian cangas.


Introduction
Convolvulaceae Juss. comprises approximately 1900 species distributed in 60 genera practically spread all over the world over a broad range of habitats, such as perennial herbs, vines, woody lianas, shrubs, or trees that are endemic to tropical regions [1][2][3][4][5][6]. In Brazil, 24 genera and approximately 420 species are recognized, occurring in various vegetation formations [7,8]. Daustinia Buril & A.R.Simões is the only genus endemic to the Flora of Brazil, where [4] transferred a Brazilian species of Jacquemontia Choisy to this new genus [4,9].
Serra dos Carajás, southeastern Amazonia, harbors approximately 30 Convolvulaceae species distributed in nine genera, with 17 of these species occurring exclusively in ironstone outcrops [10], known as canga, that is surrounded by dry and humid evergreen tropical forests [11,12]. Convolvulaceae is highly represented in canga vegetation, predominantly herbaceous and shrubby, associated with outcrops of ferruginous rocks, thus presenting a wide variety of geoenvironments, such as rocky and hydromorphic fields, as well as forest for-mations [12]. The extreme conditions in which the cangas are inserted, such as: acidic soils, poor in nutrients, in addition to high temperatures and strong seasonality, provide an environmental peculiarity, for the occurrence of a large number of endemic and rare species, among them Ipomoea cavalcantei D.F. Austin [12,13].
Indeed, even the Convolvulaceae pollen are common on bee honeys [14], lake surface sediments [15,16], and Quaternary lake cores [17,18]. However, these studies unfortunately made a genus-level identification. This makes it difficult to develop accurate pollen interaction networks based on floral visitors. Convolvulaceae pollen in sediments have also been generally associated with canga vegetation and dry environment conditions along the Quaternary, ignoring the possible relationship with humid and forest environments as currently observed.
The pollen morphology of the Convolvulaceae has been analyzed by several researchers as an important taxonomic tool [19][20][21][22][23][24][25]. Nevertheless, palynological studies in South America are scarce [26,27]. Convolvulaceae is considered to be eurypalynous [28], with a classification based on a single character, which has caused uncertainty in its taxonomic classification [29]. Therefore, this study aims to present the detailed pollen morphology of Convolvulaceae species from the canga vegetation of Serra dos Carajás, based on light and scanning electron microscopy, to evaluate the potential of distinguishing their lower taxonomic levels and habitat types, which will improve the future studies on insect-plant interaction networks as well as paleoenvironmental analyses.

Study Area
Serra dos Carajás, located in the southeastern Amazonia, comprises the largest mineral province in Brazil and one of the largest in the world. In addition, this region is also home to a huge mosaic of conservation units protected by Brazilian legislation, which has protected the Amazon rainforest from conversion to pasture over the last fifty years ( Figure 1A).
The cangas are areas with high species richness and unique floristic composition, including several endemic species that make the Carajás region an important area for the conservation of Amazonian flora [12].
The regional climate is tropical monsoon (Am; [35]). The average annual temperature is 26 • C. The rainfall regime is characterized by well-defined rainy (November to May) and dry (June to October) seasons with total annual rainfall of approximately 1700 and 240 mm, respectively [36].

Samples Collection and Slide Preparation for Morphological Descriptions
Occurrence of Convolvulaceae species at the study site is shown in Figure 1 and Table  S1, and the examined specimens are stored in the herbaria of the Museu Paraense Emílio Goeldi (MPEG) and Parque Zoobotânico de Carajás (HCJS). Flower buds (mature) were extracted from the exsicatae collections and treated using standard pollen preparation methods including flower buds fixation in acetic acid, and acetolysis [38]. All slides were deposited in the Palinoteca of the Instituto Tecnológico Vale (PALIITV). For light microscopy (LM), the pollen was mounted in glycerol jelly, examined, measured, and photographed using a Zeiss AXIO Imager M2 microscope with a Pan-APOCHROMAT 20×, 40× and 100× objective. For scanning electron microspcopy (SEM), pollen grains were dehydrated with acetone, mounted on SEM stubs, coated with gold and imaged with a Zeiss Sigma VP microscope at 2000×, 6000×, 12,000× and 20,000× magnification. The following morphological parameters were measured: grain diameter (GDL and GDW), excluding echinae in Ipomoea; for the pores, the largest and smallest diameters (Pores_length and Pores_width) and distance between them (C_pores) were calculated. For the echinae, the base (Width_base_echinae) and height (Height_echinae), as well as the distance between them (DE) were measured. Measurement of exine stratification, composed of nexine (Nexine_thickness) and sexine (Sexine), was obtained at the interechinae region. The estimation of the number of echinae (Equation (1)) and pores per pollen grain (X_echinae and X_porus; Equation (2)) was based on [39]. These variables were examined in 20 grains per sample [40].

Samples Collection and Slide Preparation for Morphological Descriptions
Occurrence of Convolvulaceae species at the study site is shown in Figure 1 and Table  S1, and the examined specimens are stored in the herbaria of the Museu Paraense Emílio Goeldi (MPEG) and Parque Zoobotânico de Carajás (HCJS). Flower buds (mature) were extracted from the exsicatae collections and treated using standard pollen preparation methods including flower buds fixation in acetic acid, and acetolysis [38]. All slides were deposited in the Palinoteca of the Instituto Tecnológico Vale (PALIITV). For light  Table S1.
For the description of pollen morphology, the terminology used followed [41]. In the morphometric description, the data were organized in the following sequence: minimum value (standard deviation) and maximum value. For echinae, the classification was adapted from [42]: conical; bulbous with apiculate apex (type 1); bulbous with rounded apex (type 2); and bulbous with bulbous apex (type 3).
The first three principal components with eigenvalues greater than 1 were considered. The results were presented in a biplot along the PC1 and PC2 axes. All analyses were performed using the statistical software R version 4.0.1 [43], and graphs were generated using 'factoextra' [44] and 'corrplot' [45].
Ipomoea L. Figures 2Q-T, 3A-T and 4A-L Pollen grains are monad, ranging from large to very large, with radial symmetry, apolar, circular, and pantoporate. The number of pores varies greatly among species ranging from~93 to~217, and they are circular to elliptical. Tectate to semitectate, columellate, and nexine is sometimes thinner than sexine, as in Ipomoea goyazensis. In all species, the macroornamentation is echinate, varying according to the echinae types ( Table 1). The microornamentation is microreticulate in the interechinae areas, with the presence of granules ( Figures 2Q and 3D,T). In I. hederifolia, echinae are evenly distributed around the pores, forming rosettes ( Figure 3Q-T). Therefore, this genus may be considered stenopalynous.

Statistical Analysis
As observed on morphological analysis, the Ipomoea species are stenopalynous. In this case, an exploratory analysis of quantitative data based on the Principal Components Analysis (PCA) was used to analyze the pollen grains ( Figure 5). This analysis was performed with 17 morphometric variables measured ( Table 2). The first three axes of the analysis, with eigenvalues greater than 1, summarized 65% of the total data variance (Table S2).

Statistical Analysis
As observed on morphological analysis, the Ipomoea species are stenopalynous. In this case, an exploratory analysis of quantitative data based on the Principal Components Analysis (PCA) was used to analyze the pollen grains ( Figure 5). This analysis was performed with 17 morphometric variables measured ( Table 2). The first three axes of the analysis, with eigenvalues greater than 1, summarized 65% of the total data variance (Table S2). The first major axis (PC1) was the most significant for species ordination ( Figure 5), which explained 30.6% of the variation based mainly on the estimate of the number of pores (X._pores) and number of echinae (X._echinae), followed by the ratios of the distance between the pori and the largest grain size and smallest grain size (C.GDL and C.GDW), and the distance between pores (C. pores). PC2 was responsible for 24.17% of the data variability, mainly related to the grain size (GDL and GDW), exine thickness, and echinae height (Height_echinae). However, echinae height and width contributed negatively to the construction of the third axis (PC3), explaining 10.25% of the data variability.
In the first axis ( Figure 5), Ipomoea cavalcantei has a larger distance between echinae (DE) values and a lower number of echinae and pores (X._echinae and X._pores). I. procumbens also has larger pores (Pl and Pw), distance between pores, and echinae base. The species I. carajasensis, I. asplundii, and I. setifera were grouped on the positive side of axes one and two, since they have higher echinae heights (Height_echinae), smaller exines and echinae bases (Width_echinae), and smaller pollen grains (GDL and GDW). In the second The first major axis (PC1) was the most significant for species ordination ( Figure 5), which explained 30.6% of the variation based mainly on the estimate of the number of pores (X._pores) and number of echinae (X._echinae), followed by the ratios of the distance between the pori and the largest grain size and smallest grain size (C.GDL and C.GDW), and the distance between pores (C. pores). PC2 was responsible for 24.17% of the data variability, mainly related to the grain size (GDL and GDW), exine thickness, and echinae height (Height_echinae). However, echinae height and width contributed negatively to the construction of the third axis (PC3), explaining 10.25% of the data variability.
In the first axis ( Figure 5), Ipomoea cavalcantei has a larger distance between echinae (DE) values and a lower number of echinae and pores (X._echinae and X._pores). I. procumbens also has larger pores (Pl and Pw), distance between pores, and echinae base. The species I. carajasensis, I. asplundii, and I. setifera were grouped on the positive side of axes one and two, since they have higher echinae heights (Height_echinae), smaller exines and echinae bases (Width_echinae), and smaller pollen grains (GDL and GDW). In the second axis, I. hederifolia was grouped separately from the other species, with the main characteristic being the largest grains among species (GDL and GDW), with smaller pores (P and Pw) and higher exine, nexine, and sexine values. In addition, I. hederifolia had a higher number of echinae and pori calculated according to [38].
The pollen of I. procumbens, I. setifera, I. asplundii, I. carajasensis, I. goyazensis, I. decora, and I. marabaensis were clustered due to similar values of the Pl.Pw ratio and Height_echinae metrics ( Figure 5).

Palynotaxonomy
Among the species studied, Cuscuta insquamata and Evolvulus felipes have the smallest pollen grains, which may be related, but without generalizations, to the size of their flowers [50]. This morphological information can approximate the species and help in the taxonomic delimitation, because until the present study little was known about the Cuscuta [51]. Evolvulus filipes was described as pantocolpate with microechinate ornamentation on SEM, which corroborates with the descriptions of [24,55].
Considering the grouping of different pollen morphology characters, ref. [21] divided Convolvulaceae into four groups, as follows: Group 1, 5(-6)-colpate; Group 2, 3colpate; Group 3, dodecacolpate; Group 4, pantoporate. According to this classification, Cuscuta insquamata belong to Group 2, which is characterized by medium-sized colpate pollen grains and the presence of granules inside the colpi. The Ipomoea species belongs to Group 4, due to the apertural type and its surface arrangement in the grains.
The echinae of Ipomoea are supported by thick columellae, which increase in height in the aperture-echinae direction, similar to the description by [24,52].
Echinae have been related as the main differentiating character of the Ipomoea species, defined according the base of the echinae, which ranges from straight to bulbar, and apex shape [47]. Therefore, they can be grouped in this study as follows: (a) conical (I. asplundii, I. carajasensis, I. procumbens, and I. setifera); (b) bulbous type 1, with bulbar base and apiculate apex (I. goyazensis); (c) bulbous type 2, with bulbar base and rounded apex (I. decora, I. hederifolia and I. marabaensis); d) bulbous type 3, with bulbous apex (I. cavalcantei). The sexine was thinner than the nexine in I. hederifolia, I. marabaensis, I. procumbens, and I. setifera. In addition, it presented the largest pollen grains with the largest diameter, ranging from 131-144 µm, classified as very large, corroborating with [50]. However, I. hederifolia does not fit with the data presented here, as they described the echinae as conical and located on edges, with bulbous echinae type 2 [53].
Intraspecific variations are observed, hindering the standardization of the morphological description, and are better detected in some species, such as I. cavalcantei and I. marabaensis. Likewise, it is difficult to establish the number of pores due to the density of the echinae and thick exine. The large number of apertures is possibly associated with derived taxa, and with greater reproductive efficiency due to increased opportunities for pollen tube germination [56].
Few diagnostic characters are known for Jacquemontia, resulting in identifications that are, in many cases, inaccurate. This is reflected in several botanical collections where the genus is erroneously identified as Evolvulus L. or Convolvulus L. [57,58]. However, the number of apertures and their distribution in the pollen grains helped in the differentiation of Jacquemontia tamnifolia and Evolvulus filipes.
New taxonomic combinations were presented by [59], with Distimake Raf. (=Merremia Dennst. ex Endl.). Due to this nomenclatural change based mainly on the phylogeny of the group, many palynological works prior to publication treated the species occurring in the cangas of Carajás as Merremia macrocalyx (Ruiz & Pav.) O'Donell.
Distimake macrocalyx has been erroneously described with psilate ornamentation [46]. In the SEM analysis, granulate and microreticulate exines were observed [60], which were classified as granules by [42]. D. macrocalyx has been described as having distally branched columellae [22]. The main differences between D. macrocalyx and Operculina hamiltonii are the size and shape, where D. macrocalyx has large and subprolate pollen grains, while O. hamiltonii has very large pollen grains with a spheroidal prolate shape. These findings corroborate with [61] and [42], but differ from those findings reported by [62], specifically related to D. macrocalyx (prolate grains). According to [26], 4-colpate pollen grains are found in D. macrocalyx, a characteristic that was not observed in the studied grains.
This study indicated that the qualitative characteristics of the echinae type, and in some cases, grain size and aperture arrangement, are important characteristics to describe the Convolvulaceae genera, thus establishing the classification of pollen types for the analyzed species. In addition, quantitative data (morphometry) confirm that the attributes used are suitable to classify the pollen types. Statistical methods such as PCA have been frequently used to evaluate the systematic utility of pollen data [63][64][65][66][67][68][69][70]. For example, I. goyazensis and I. decora presented conflicts in their taxonomic delimitations [71]. Indeed, we observed based on the PCA that the two species were grouped together ( Figure 5), which may serve as evidence of their similarity.
The studied specimens of the genus Ipomoea were collected in different areas, which suggests that the geographical boundaries were not sufficient to result in a significant difference in pollen morphology, with the exception of I. hederifolia, which was grouped separately from the others. According to [72], closely related species generally produce similar pollen grains. Some specimens, represented by same-color points on the PCA plot ( Figure 5), are dispersed in the cluster, differing in the number of apertures in their pollen grains, such as I. carajasensis. This trend is often due to hybridization processes, and is linked to the level of ploidy in individuals [73][74][75].

Relevance for Interaction Networks and Paleoenvironmental Studies
The Convolvulaceae occurrence in Carajás cangas were based on a huge botanical survey and re-analysis of exsiccate by family specialists conducted by the Flora de Carajás Project [10,11]. Therefore, the family may be found in a wide variety of canga geoenvironments with multiple flower visitors [10,33].
Their flowers generally attract a wide range of visitors that include bees (melittophily) as the predominant group [76]. Indeed, from the studied species, only Cuscuta insquamata, Ipomoea cavalcantei, and Ipomoea hederifolia are ornithophilous [77]. This is a very interesting field observation for C. insquamata, since Cuscuta species usually have a large and varied court of visiting insects, including flies, moths, beetles, and predators such as spiders and larger insects [78]. Ipomoea flowers are tubular and should restrict entry into the floral tube only to visitors with adequate anatomy, thus characterizing a relationship between the size of the floral tube and the size of the visitor resulting in contact with reproductive structures, as a way to guarantee pollination [79]. Among the floral visitors of Ipomoea species, Melitoma bees are usually associated with their flowers [79][80][81][82]. Trigona bees are legitimate frequent visitors on I. cavalcantei and I. marabaensis. However, the predominant pollinators of I. cavalcantei are hummingbirds, according to [83]. The exine ornamentation in relation to pollinators has been described in reference [84]. Several plant species with microreticulated pollen grains are pollinated by bees [85][86][87]. This may be the case for Aniseia martinicensis, C. insquamata, Evolulus filipes, Jacquemontia tamnifolia, Distimake macrocalyx, and Operculina hamiltonii. The echinate surface is also a typical feature for pollen transfer by animals [84]. Indeed, echinae in Ipomoea pollen seem to allow pollen fixation to the hair of bees, optimizing the transport process [60]. Therefore, the pollen morphology is one of the factors responsible for the strengthening of the plant-pollinator relationship [88]. Convolvulaceae pollen in sediments were only identified at the genus level (i.e., Ipomoea and Evolulus), and were generally associated with canga vegetation and dry environment conditions along the Quaternary, ignoring the possible relationship with humid and forest environments, as currently observed [17,18,88]. In fact, this generalization is no longer acceptable. Aniseia martinicensis, C. insquamata, Distimake macrocalyx, Evolvulus filipes, I. asplundii, I. carajasensis, I. cavalcantei, I. decora, I. goyazensis, I. hederifolia, I. marabaensis, I. procumbens, I. setifera, Jacquemontia tamnifolia and Operculina hamiltonii are not exclusively found on rupestrian fields (Fe-Al lateritic crusts), but are also very common on swampy fields and borders of humid evergreen tropical forests (HETF), and dry and open forests, which suggests more humid habitat conditions and some protection from direct sunlight. In addition, the low classification level of Ipomoea and even Jacquemontia hinders the possible association with anthropic interventions in the natural environment. Indeed, I. hederifolia, I. setifera, and J. tamnifolia are frequently observed in altered areas as ruderal species [10].

Conclusions
These results reinforce the importance of studying pollen morphology to identify and distinguish genera and species of the family Convolvulaceae. Based on the data obtained, Aniseia martinicensis has large pantocolpate pollen with microreticulate/microverrucate ornamentation. A similar apertural type was found in Evolvulus filipes, but with medium-sized grains with microreticulate/microechinate ornamentation. Cuscuta insquamata, Distimake macrocalyx, and Operculina hamiltonii have 3-colpate pollen grains, but C. insquamata has medium-sized grains with microreticulate and microechinate ornamentation, while D. macrocalyx and O. hamiltonii have subprolate and prolate spheroidal grains, respectively. Ipomoea are pantoporate with echinate ornamentation. Indeed, echinae are the main differentiating character of the Ipomoea species, defined according to their basal morphology. However, apertural type and grain size must be used in conjunction to better classify each species. I. asplundii, I. carajasensis, I. procumbens, and I. setifera have conical echinae with 163, 183, and 144 pores, respectively. I. goyazensis present bulbous type 1 echinae. I. decora, I. hederifolia, and I. marabaensis have type 2 bulbous echinae with~160, 217, and 182 pores. I. cavalcantei has bulbous type 3 echinae. It is concluded that palynotaxonomy is considered an important and effective tool in species identification for taxonomic studies. This study of the Convolvulaceae taxa contributes to the knowledge on the Brazilian and worldwide pollen flora, and may contribute to taxonomic circumscription, and thus improve the understanding of the phylogenetic relationships of Convolvulaceae. In general, the set of morphological characters was effective for separating Convolvulaceae genera and species occurring in Serra dos Carajás and can be a reliable tool for future studies on insect-plant interaction networks as well as paleoenvironmental analyses.